Team:UChicago/Measurements

One of the most
significant design choices in our oscillator plasmid was the inducible
L-rhamnose promoter (pRha). We based our choice by researching the
characterization of a variety of different inducible promoters in the iGEM
registry. The reason we chose this specific L-rhamnose inducible promoter,
compared to a more standard IPTG inducible promoter was because the
characterization for the L-rhamnose inducible promoter (Part:BBa_K914003),
portrayed a more well characterized tight and graded response of pRha by the
2012 Paris-Bettencourt team. This leads us to believe that pRha is more suited
to fine tune the concentrations of KaiA in order to investigate the KaiA : KaiC
ratio in vivo.

Figure H: Characterization graph developed by the
2012 Paris-Bettencourt Team.

We
conducted our own characterization of this Biobrick by tracking KaiA expression
for a gradient of L-rhamnose percentages during a 10 hour induction (10 hour
induction time was reported by the 2012 Paris-Bettencourt team). In order to
track KaiA expression we used western blotting techniques as well as protein
densitometry analysis in Image J. We believe our characterization is an
improvement on the characterization accomplished by the 2012 Paris-Bettencourt
team which used a plate-reader.

The results from our western
blots indicate the tightness of pRha. At 0% L-rhamnose, there is significantly
less KaiA expressed compared to KaiB and KaiC. This supports our ability to
tune KaiA concentrations to optimize oscillations.

The gradient curve we
developed portrays the ability to fine tune the concentrations of KaiA. The
data in this graph was corrected using ng from protein standards (Figure J).
Ideally, we would have run multiple such assays, with a higher gradient of
L-rhamnose than listed above. However, the data presented gives us a good start
to characterizing a usable gradient of KaiA expression. Given our previous
data, in which KaiC levels varied from a range of 1.6-3.7 ngs, we can ballpark
the %Rhamnose to investigate to be from 1-0.0001%.